Isolated femurs and tibia were flushed with PBS, and precursor cells were cultured in complete RPMI supplemented with 30% L929 cell-conditioned medium. After 7 d in culture, mature bone marrow-derived macrophages (BMDM) were harvested and infected with metacyclic promastigotes at different multiplicities of infection (MOI). For detection of IL-1β secretion by infected cells by ELISA, BMDMs were primed for 5 h with LPS (50 ng/ml), washed, and then infected with metacyclic promastigotes at different MOIs for 6 hr. Supernatants were assayed using the IL-1β DuoSet ELISA (R&D Systems). For western blot analysis of the active form of IL-1β, BMDMs were treated or not with LPS (50 ng/ml) and infected or not with metacyclic promastigotes at a 1:8 MOI for 6 hr. Supernatants were concentrated by methanol/chloroform precipitation and immunoblotted along with cell extracts for IL-1β (AF-401/R&D) and caspase-1 (M-20/Santa Cruz Biotech). For selective inhibition of caspase-1, BMDMs were pretreated with 20μM and 50 μM of Z-WEHD-FMK or control inhibitor Z-FA-FMK (R&D Systems) for 5 hr prior to infection.
Af 401
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The AF-401 is a laboratory equipment product developed by R&D Systems. It is designed to perform specific functions within the research and development laboratory environment. The core function of the AF-401 is to [DESCRIPTION_NOT_AVAILABLE].
Automatically generated - may contain errors
Lab products found in correlation
Ag 20b 0014, by Adipogen (3 mentions)
β actin, by Merck Group (3 mentions)
Epr16883, by Abcam (3 mentions)
G box chemi xx6, by Syngene (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Cd11b magnetic bead, by Miltenyi Biotec (2 mentions)
Amersham ecl detection reagent, by GE Healthcare (2 mentions)
Asc d2w8u, by Cell Signaling Technology (2 mentions)
Cryo 2, by Adipogen (2 mentions)
Ab53219, by Abcam (2 mentions)
Anti mhcii, by BioLegend (2 mentions)
Anti f4 80 123120, by BioLegend (2 mentions)
Shp 1, by Cell Signaling Technology (2 mentions)
Af 400, by Cell Signaling Technology (2 mentions)
Myosin 9, by Cell Signaling Technology (2 mentions)
P42 44 mapk erk1 2, by Cell Signaling Technology (2 mentions)
Phospho mlkl, by Abcam (2 mentions)
Phospho p38 mapk t180 y182, by Cell Signaling Technology (2 mentions)
Ripk1, by Cell Signaling Technology (2 mentions)
P38 mapk, by Cell Signaling Technology (2 mentions)
15 protocols using af 401
1
Leishmania Infection of Bone Marrow-Derived Macrophages
2
Immunoblotting Antibodies for Inflammation
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The following antibodies for immunoblotting were used. Anti-β-actin-HRP (sc-47778, Santa Cruz Biotechnology or A3854, Sigma-Aldrich), goat anti-mouse IL-1β (AF-401, R&D systems), anti-mouse caspase-1 (Casper 1, AG-20B-0042-C100, Adipogen), anti-ASC (AG-25B-0006, Adipogen) and anti-NLRP3 (AG-20B-0014, Adipogen). Secondary anti-mouse and anti-goat antibodies were obtained from Santa Cruz Biotechnology, anti-rabbit secondary antibody was from Thermo Fisher Scientific.
3
Immunoblot Analysis of Inflammatory Proteins
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Cells were lysed in 5× sample buffer and separated by SDS-PAGE and blotted according to standard protocols. For measurement of cleaved IL-1β and caspase-1 in the supernatant, proteins in the supernatant were precipitated with 1% (v/v) StrataClean resin (Agilent Technologies), and beads were lysed in 5× sample buffer. Proteins were visualized using the HRP substrate WesternBright ECL spray (Advansta) on a ChemiDoc imaging system (Bio-Rad), apart from blots presented in Figs. 2 b and 3e , which were developed using traditional X-ray film. All uncropped and unprocessed scans of the most important blots are available in the Source Data file. Primary Abs were β-actin (1:15,000, AC-74, Sigma-Aldrich), Gapdh (1:5000, 6C5, Calbiochem), IL-1β (pro- and cleaved, 1:1000, AF-401, R&D Systems), caspase-11 (1:1000, 17D9, Sigma-Aldrich), p-STAT-1 (1:1000, #9177, Ser727, Cell Signalling) and total STAT-1 (1:1000, #9172, Cell Signalling), and caspase-4 (4B9, MBL). HRP-conjugated secondary Abs were from Jackson ImmunoResearch Laboratories.
4
Microglia Characterization After Stroke
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Littermate WT and NLRP3−/− mice were transcardially perfused with saline 24 hours after stroke. Brains were then removed and digested into a single-cell suspension and myelin removed (as described above), and cells were isolated by magnetic-activated cell sorting using CD11b+ magnetic beads (Miltenyi). Positive and negative CD11b+ cells were lysed in Tris-Triton buffer (150 mmol/L NaCl, 1% [vol/vol] Triton X-100, 50 mmol/L Tris, pH 7.5), supplemented with a protease inhibitor mixture. Cell lysates were separated by Tris-glycine SDS/PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes at 25 V using a semidry Trans-Blot Turbo system (Bio-Rad). Membranes were blocked in 2.5% (wt/vol) BSA in PBS, 1% (vol/vol) Tween 20 before being incubated with indicated primary antibodies at 4°C overnight. Membranes were then labeled with horseradish peroxidase–tagged secondary antibodies and visualized with Amersham ECL detection reagent (GE Healthcare). Western blot images were captured digitally using a G:Box Chemi XX6 (Syngene). Specific antibodies were used targeting mouse IL-1β (AF-401; R&D), ASC (D2W8U, Cell Signalling Technology), caspase-1 p10 (EPR16883; Abcam), and β-actin (Sigma).
5
Neutrophil Degranulation Protein Analysis
6
Western Blot Analysis of Synovial Tissue Proteins
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The protein concentration of the synovial tissue extracts (L) was determined in the clarified supernatants using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were electrophoresed on Mini-PROTEAN TGX 4–20% gels (Bio-Rad Laboratories) and transferred to nitrocellulose of 0.45 μm pore size (GE Healthcare Life Sciences, Marlborough, MA, USA). Membranes were blocked in 5% dried milk in PBS-Tween 0.5% for 1 hour at room temperature. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (JNK) (1:500; Cell Signaling Technology, Danvers, MA, USA), IL-1β (1:1000, AF-401; R&D Systems), and NLRP3 (1:1000, Cryo-2; AdipoGen Life Sciences, San Diego, CA, USA) were used as the primary antibodies. Peroxidase-conjugated secondary antibodies and chemiluminescence were used to develop the blots. A primary antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) was used to assess protein loading.
7
Aorta Histological Analysis in Mice
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Mice were perfused with PBS under physiologic pressure and aortas were harvested. Tissues were embedded in O.C.T. compound (Sakura Tissue Tek, Alphen aan den Rijn, the Netherlands) and sectioned in 6 μm thickness using Leica CM3050S cryostat. Hematoxylin and eosin, Verhoeff-Van-Gieson, and Masson trichrome stainings were conducted. Immunostaining was also performed according to standard protocols with the following antibodies: anti-CD206 (AF2535, R&D Systems, Minneapolis, Minn), anti-F4/80 (123120, Biolegend, San Diego, Calif), anti-MHCII (107616, Biolegend), anti-MYH11 (ab53219, Abcam, Cambridge, UK), and anti-IL-1β (AF-401, R&D Systems). A total of three sections, approximately 60 μm apart, were stained and analyzed for each mouse aorta. The integrated density (the sum of the value of the pixels in the image or selection), area of immunofluorescence (square pixels), and number of signal positive cells were measured using ImageJ Software (National Institutes of Health, Bethesda, Md), as previously described.16 (link)
8
Neutrophil Lysis and Protein Analysis
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Neutrophils were lysed in 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 mM NaF, 1 mM sodium vanadate, 1 mM phenylmethanesulphonylfluoride in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and Complete Protease Inhibitor Cocktail (Roche). Cell lysates and supernatants were analyzed using antibodies specific to IL-1β/IL-1F2 (R&D; AF-401), IL-1α/IL-1F1 (R&D; AF-400), RIPK1 (Cell Signaling Technology; D94C12), SHP-1 (Cell Signaling Technology; C14H6), p38 MAPK (Cell Signaling Technology; D94C12), phospho-p38 MAPK (T180/Y182) (Cell Signaling Technology; 9211), Myosin-9 (Cell Signaling, #3403), p42/44 MAPK (ERK1/2) (Cell Signaling Technology; 137F5), or phospho-MLKL (S345) (Abcam; EPR9515[2]). Westerns were developed (ECL Substrate), visualized (ChemiDoc), and analyzed (ImageLab Software) with BioRad products.
9
Immunoblot Analysis of Inflammasome Components
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Precipitated supernatants and BMDMs extracts were analyzed by immunoblot as described previously (15 (link)). The primary antibodies used were anti-mouse IL-1β (AF-401, R&D, Minneapolis, MN, USA), anti-mouse caspase-1 (p20) (AG-20B-0042, Adipogen, Liestal, Switzerland), anti-ASC (AG-25B-0006-C100, Adipogen, Liestal, Switzerland), anti-NLRP3 (AG-20B-0014, Adipogen, Liestal, Switzerland), and anti-mouse β-actin (Proteintech, Wuhan, China).
10
Neospora caninum Infection in Peritoneal Macrophages
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PMs were seeded in 6-well plates at a density of 3 × 106 cells/well, and pre-treated for 1 h with NAC, then infected with N. caninum (MOI of 3:1 2:1 or 1:1). In some experiments, PMs were incubated with N. caninum for 8 h, then treated with PG at different concentrations. At 24 or 36 h post-infection, the cell lysates and supernatants were collected and assessed by western blotting, as described previously [27 (link)]. The following primary antibodies were incubated overnight at 4 °C: anti-mouse IL-1β (p17; 1/1,000; AF-401; R&D, Minneapolis, MN, USA); anti-mouse caspase-1 (p20; 1/1,000; AG-20B-0042; Adipogen, Liestal, Switzerland); anti-NLRP3 (1/1,000; AG-20B-0014, Adipogen); and anti-mouse β-actin (1/2,000; 60008–1; Proteintech, Wuhan, China). Secondary horseradish peroxidase (HRP)-conjugated rabbit anti-goat or anti-mouse IgG (1/5,000; Proteintech) was then added for 1 h at room temperature (RT). Finally, proteins were visualized using an Enhanced chemiluminescence (ECL) Western Blot Detection System (Clinx Science Instruments, Co., Ltd., Shanghai, China).
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